This invention is directed to a thermostable type I DNA topoisomerase V from the thermophilic prokaryote Methanopyrus kandleri, methods for its purification, and methods for its use.
The intertwining of the two strands of the DNA helix presents a number of topological problems which the cell must overcome in order to regenerate, recombine, and express its genetic information (M. Gellert, Annu. Rev. Biochem. 50:879-910 (1981); J. C. Wang, Annu. Rev. Biochem. 54:665-697 (1985); A. Maxwell and M. Gellert, Adv. Protein Chem. 38:68-107 (1986); N. Osheroff, Pharmac. Ther. 41: 223-241 (1989); J. C. Wang et al., Cell 62:403-406 (1990); J. C. Wang, J. Biol. Chem. 266:6659-6662 (1991); A. Kornberg and T. A. Baker, in DNA Replication (2d ed.), W. H. Freeman and Company, New York, 1992, pp. 379-401; J. C. Wang and L. F. Liu, in DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990, pp. 321-340). Consequently, the enzymes modulating the topological state of nucleic acids, known as DNA topoisomerases, play a crucial role in controlling the physiological functions of DNA.
To simplify the later description, a few definitions relevant to DNA topology are useful. Supercoiling is typical in closed circular DNA (ccDNA) because of the topological linkage of the two complementary strands. The linking number, Lk, is the quantitative measure of this linkage: it is the algebraic number of times one strand crosses the surface stretched over the other strand. The Lk value is a topological invariant for ccDNA; there is no way it can be changed without introducing chain scissions. The number of supercoils can be defined as EQU .DELTA.Lk=Lk-N/.gamma..sub.o
where N is the number of base pairs in a DNA and .gamma..sub.o is the number of base pairs per turn of the double helix under given ambient conditions. Specific linking difference, or superhelical density, is defined as EQU .sigma.=.gamma..sub.o .DELTA.Lk/N
If .DELTA.Lk&gt;0, DNA is called positively supercoiled; if .DELTA.Lk&lt;0, DNA is negatively supercoiled. A consequence of negative supercoiling is that the DNA helix is more easily unwound, i.e., the strands are more readily separated, whereas positive supercoiling, by tightening the pitch of the helix, would make unwinding more difficult.
Any changes in the Lk value are resolved into its two geometrical components (J. H. White, Am. J. Math. 91: 693-728 (1969)) EQU .DELTA.Lk=.DELTA.Tw+Wr
where .DELTA.Tw is the difference in the axial twist of either strand about the axis of the double helix, Wr is the quantity related to supercoiling and is determined by the spatial shape of the axis of the double helix (for detailed discussions on general aspect of DNA topology, see DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990)).
DNA transformations performed by DNA topoisomerases are accomplished by the cleavage of either a single strand or both strands. The unit change in the Lk upon such transformations is the best operational distinction between the two classes of topoisomerases (P.O. Brown & N. R. Cozzarelli, Science 206:1081-1083 (1979)). DNA topoisomerases whose reactions proceed via a transient single-stranded break and changing the Lk in steps of one are classified as type 1, while enzymes whose reactions proceed via double-stranded breaks and changing the Lk in steps of two are classified as type 2.
Members of type 2 topoisomerase family include DNA gyrase, bacterial DNA topoisomerase IV, T-even phage DNA topoisomerases, eukaryotic DNA topoisomerase II, and thermophilic topoisomerase II from Sulfolobus acidocaldarius (see reviews cited above; A. Kikuchi et al., Syst. Appl. Microbiol. 7:72-78 (1986); J. Kato et al., J. Biol. Chem. 267:25676-25684 (1992); W. M. Huang in DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990), pp.265-284; T.-S Hsieh in DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990), pp.243-263)). The coding sequences of a dozen or so type 2 enzymes have been determined, and the data suggest that all these enzymes are evolutionarily and structurally related. Topological reactions catalyzed by type 2 topoisomerases include introduction of negative supercoils into DNA (DNA gyrase), relaxation of supercoiled DNA, catenation (or decatenation) of duplex circles, knotting and unknotting of DNA.
The family of type 1 topoisomerases comprises bacterial topoisomerase I, E. coli topoisomerase III, S. cerevisiae topoisomerase III (R. A. Kim & J. C. Wang, J. Biol. Chem. 267:17178-17185 (1992)), the type 1 topoisomerase from chloroplasts that closely resembles bacterial enzymes (J. Siedlecki et al., Nucleic Acids Res. 11:1523-1536 (1983)), thermophilic reverse gyrases (A. Kikuchi in DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990), pp.285-298); C. Boutbier de la Tour et al. J. Bact. 173:3921-3923 (1991)), thermophilic D. amylolyticus topoisomerase III (A. I. Slesarev et al., J. Biol. Chem. 266: 12321-12328 (1991), nuclear topoisomerases I and closely related enzymes from mitochondria and poxviruses (N. Osheroff, Pharmac. Ther. 41:223-241 (1989)). With respect to the mechanism of catalysis these topoisomerases can be divided into two groups. Group A consists of enzymes that are single-strand-specific, require a divalent cation for activity, and form a transient covalent complex with the 5'-phosphoryl termini (prokaryotic type 1 topoisomerases and S. cerevisiae topoisomerase III). Group B includes type 1 topoisomerases that operate on duplex DNAs, do not require a divalent cation for activity, and bind covalently to the 3'-phosphoryl termini (nuclear topoisomerases I, enzymes from mitochondria and poxviruses commonly called eukaryotic topoisomerases I). Type 1 topoisomerases can carry out the following topological reactions; they relax supercoiled DNA (except of reverse gyrases), catenate (or decatenate) single-stranded circular DNAs or duplexes providing that at least one of the molecules contains a nick or gap, interact with single-stranded circles to introduce topological knots (type 1-group A topoisomerases). Reverse gyrase, belonging to type 1-group A topoisomerases, is the only topoisomerase shown to be able to introduce positive supercoils into ccDNA.
Research on DNA topoisomerases has progressed from DNA enzymology into developmental therapeutics. Bacterial DNA topoisomerase II is an important therapeutic target of quinolone antibiotics; mammalian DNA topoisomerase II is the cellular target of many potent antitumor drugs (K. Drlica, Microbiol. Rev. 48:273-289 (1984) and Biochemistry 27: 2253-2259 (1988); B. S. Glisson & W. E. Ross, Pharmacol. Ther. 32:89-106 (1987); A. L. Bodley & L. F. Liu, Biotechnology 6: 1315-1319 (1988); L. F. Liu, Annu. Rev. Biochem. 58:351-375 (1989)). These drugs, referred to as topoisomerase II poisons, interfere with the breakage-rejoining reaction of type II topoisomerases by trapping a key covalent reaction intermediate, termed the cleavable complex. Mammalian type 1-group B topoisomerase has been shown to be the target of camptothecin (CPT), a plant alkaloid with strong antitumor activity. CPT and its derivatives also trap a putative covalent reaction intermediate, the clearable complex. This type of reversible DNA damage is lethal to proliferating cells and is responsible for the antitumor activity of topoisomerase poisons (Y.-H. Hsiang et al., J. Biol. Chem. 260:14873-14878 (1985); Y. Hsiang et al., Cancer Res. 49: 5077-5082 (1989); C. Holm et al., Cancer Res. 49:6365-6368 (1989), P. D'Arpa et al. Cancer Res. 50:6919-6924 (1990); A. Y. Chen et al. Cancer Res. 51:6039-6044 (1991)). Increased attention to CPT and its derivatives as the most promising anticancer agents currently in clinical trials (W. J. Slichenmyer et al., J. Natl. Cancer Inst. 85:271-287 (1993); U.S. Pat. No. 5,106,742 (camptothecin analogues as potent inhibitors of topoisomerase I); U.S. Pat. No. 5,122,526 (camptothecin and analogues thereof and pharmaceutical compositions and method using them)) resulted in isolation several CPT-resistant cell lines and a CPT-resistant mutant of human topoisomerase I has been characterized (H. Tamura et al., Nucleic Acids Res. 19:69-75 (1991)). Through the use of a yeast strain in which yeast type 1-group B topoisomerase is replaced by its human counterpart, other CPT-resistant mutants of the human enzyme have been isolated (P. Benedetti et al., in Drug Resistance as a Biochemical Target in Cancer Chemotherapy (T. Tsuruo, M. Ogawa, & S. K. Carter, eds., Academic Press, San Diego, Calif., 129, 1991)). Further identification of the mutation sites of CPT-resistant type 1-group B topoisomerases is potentially useful for modeling novel derivatives acting against CPT-resistant tumor cells.
To date type 1-group B topoisomerases have been found only in eukaryotes. One might expect that the finding of a prokaryotic counterparts to eukaryotic type 1-group B topoisomerases would be viewed with great interest by pharmacologists and clinicians as well. Whereas it would be important to exploit the common features of type 1-group B topoisomerases, it would be equally important to exploit the differences among them for modeling novel drugs. Also the new organisms harboring type 1-group B topoisomerases would be of clinical interest as potential sources of the natural inhibitors of those enzymes. For example, in E. coli, the miniF plasmid CcdB protein, like quinolone antibiotics and antitumoral drugs, promotes DNA gyrase-mediated double-stranded DNA breakage (P. Bernard & M. Couturier, J. Mol. Biol. 226:735-745 (1992)).
Another aspect of medical utility of type 1-group B topoisomerases is the identification of the human Scl-70 antigen as DNA topoisomerase I. Scleroderma (progressive systemic sclerosis) patients may produce high titer autoimmune antibody directed against human topoisomerase I (J. H. Shero et al., Science 231:737-740 (1986)). The availability of cloned human topoisomerase I enables the development of methods for rapid screening for the presence of these autoantibody in patient sera (P. D'Arpa et al. Proc. Natl. Acad. Sci. USA 85:2543-2547 (1988); U.S. Pat. No. 5,070,192 (cloned human topoisomerase I: cDNA expression and use for autoantibody detection)). Remarkably, type 1-group B topoisomerases of higher plants are recognized by human anti-topoisomerase autoantibody, despite the divergence of the kingdoms (P. F. Agris et al., Exp. Cell Res.189:276-279 (1990)). It is conceivable that human autoantibody could recognize type 1-group B topoisomerases from prokaryotic organisms as well. Using human antibody on prokaryotic systems immunoprecipitations, competitive binding assays, cellular function studies, and probing expression libraries could be accomplished. The use of human anti-topoisomerase antibody as probes of prokaryotic type 1-group B topoisomerase structure may be important in further understanding the interaction of human topoisomerase I and cancer chemotherapeutic agents. Finally, such cross-reactivity would have clinical significance for autoimmunity as well. Sera of scleroderma patients targeted at least 6 independent epitopes on human topoisomerase I. Molecules of cDNA comprising a part of the cDNA sequence of human topoisomerase I which encode at least one epitope for autoantibody to human topoisomerase I are available (P. D'Arpa et al., Arthritis Rheum. 33:1501-1511 (1990)). Other type 1-group B topoisomerases reacting with anti-human topoisomerase I antibody could be used in the same way. It would be important in tracking the emergence of autoimmune antibody against particular epitopes during the progression of disease.
One of the driving forces behind molecular biology is the successful utilization of enzymes as reagents. Mesophilic type 1-group B topoisomerases are widely used for analysis of DNA supercoiling, DNA conformation, transcription in vitro, and chromatin reconstitution (M. D. Frank-Kamenetskii, in DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990), pp.185-215; Y. Zivanovic et al., J. Mol. Biol. 214:479-495)). The availability of thermophilic type 1-group B topoisomerases simplifies significantly the manipulation of DNA conformation. Reverse gyrase is becoming an indispensable tool for preparation of positively supercoiled DNA, while D. amylolyticus topoisomerase III is a convenient tool for preparation of ccDNA with different degree of negative supercoiling (A. I. Slesarev et al. J. Biol. Chem. 266: 12321-12328 (1991)).
Chromatin reconstitution in vitro is usually done at 0.5-1.0 M while eukaryotic topoisomerase I is inhibited by 0.2 M and higher concentration of NaCl (P. A. Der Garabedian et al., Biochemistry 30:9940-9947 (1990)). At 0.2 M NaCl nucleosome assembly is very inefficient, so there is a need in topoisomerase working in a wide range of ionic strength. Moreover, recent progress in this field increases the need for thermostable topoisomerases with properties akin to those of eukaryotic topoisomerase I (J. Bashkin et al. Biochemistry, 32:1895-1898 (1993)).
Other applications of topoisomerases may exploit their ability to cleave DNA at specific sites ("specific endonucleases"), their ability form a covalent complex with DNA, their substrate selectivity. Of potential interest is a topoisomerase religation half-reaction. Type 1-group B topoisomerases catalyze the in vitro ligation of nonhomologous DNA fragments lacking any sequence homology or complementarity (M. D. Been & J. J. Champoux, Proc. Natl. Acad. Sci. USA 78:2883-2887 (1981)). Pan et al. disclosed a method for ligation of artificial substrates that bear a tyrosine residue on the 3'-PO.sub.4 of an appropriate oligonucleotide with mammalian topoisomerase I (J. Biol. Chem. 268:3683-3689 (1993)).
Preparation and analysis of specific nucleotide and protein sequences constitutes a basis of current molecular biology, biotechnology, and molecular medicine. Enzymatic DNA sequencing (F. Sanger, Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) using Sequenase enzyme (US Biochemical, Cleveland, Ohio) is currently the most popular method to obtain genetic information. Any improvement in the procedure that will eliminate labor-consuming steps or increase sensitivity, is of great demand in the field. It is generally accepted that, despite problems with DNA denaturation and reassociation, the use of double-stranded DNA for sequencing is preferable to the construction and use of single-stranded DNA. The denaturation of conventionally purified ccDNA involves alkali treatment that is hazardous for DNA followed by time and labor-consuming DNA precipitation, during which reassociation can occur (S. M. Adams et al. Focus 13:56-58 (1991); D. F. Barker, Biotechniques 14:168-169 (1993)). This results in fewer readable bases. Sequencing at higher temperatures can eliminate reassociation, but results in a higher error rate.
Thus, there is a need for a reliable technology of plasmid template preparation for sequencing compatible with popular sequencing protocols.
Polymerase chain reaction (R. K. Saiki et al., Science 230:1350-1354 (1985); H. A. Erlich, ed., PCR technology: Principles and applications for DNA amplification (Stockton Press, New York, 1989)) is the basic technique that allows amplification of pieces of DNA starting from tiny amounts of material with limited knowledge about its exact primary structure. Its development and refinement continue at a rapid pace. Another chain reaction introduced in 1990 (European Patent Application EP 88311741.8; F. Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1990)) is based on ligase and is becoming a powerful tool in medical diagnostics. The success of these techniques was due to the use of thermostable enzymes. However, when PCR or LCR amplification is performed on a plasmid template, poor denaturation of ccDNA may result in a failure to detect products (E. C. Lau et al., Biotechniques 14:378 (1993)). Therefore, these techniques also need a procedure of plasmid template preparation for reliable primer annealing.
One of the solutions of the above problem consists in the substitution of DNA denaturation with a special and simple topological treatment with a thermostable enzyme(s) that will allow a primer to anneal to the double-stranded DNA with same efficiency as to the single-stranded DNA. The effect is based on the topological destabilization (unlinking) of the double helix that can be varied in a wide range. The limit products of the unlinking are ccDNA with a few links or even single-stranded complementary rings. Such DNA, called form V, can contain 10 to 40% single-stranded regions at room temperature and will melt easily at elevated temperature (U. H. Stettler et al., J. Mol. Biol. 131:21-40 (1979)). The non-template strand of the duplex will not hinder subsequent elongation and moreover it will create a topological force in favor of elongation. The method is equally applicable to any procedure that involve primer annealing and/or elongation, i.e., enzymatic ccDNA sequencing, PCR, LCR, hybridization probe preparation, etc.
There are several enzymatic procedures for preparation of highly underwound ccDNA molecules (M. Iwabuchi et al., J. Biol. Chem. 258:12394-12404 (1983); A. M. Wu et al., Proc. Natl. Acad. Sci. USA 80:1256-1260 (1983); T. A. Baker et al., Cell 45:53-64 (1986); B. F. Pugh & M. M. Cox, J. Biol. Chem. 262:1326-1336 and 1337-1343 (1987); C. A. Parada & K. J. Marians, J. Biol. Chem. 264: 15120-15129 (1989); B. F. Pugh et al., J. Mol. Biol. 205: 487-492 (1989)). However, the procedures require multienzyme complexes of at least two different enzymes and specific buffer conditions. In addition, the enzymes used in the above procedures are thermolabile and can not be used in PCR or LCR.
Slesarev et al. disclosed that thermostable type 1-group A topoisomerase III from Desulfurococcus amylolyticus can alone substantially reduce the Lk value of ccDNA and generate highly unwound forms of ccDNA in the linear DNA melting range (J. Biol. Chem. 266:12321-12328 (1991)). However, this topoisomerase is Mg.sup.2+ -dependent, ineffective at low salt conditions, and inhibited by single-stranded DNA. In addition, it needs very high temperature to be active on relaxed and positively supercoiled DNA. These properties of Dam topoisomerase III will interfere with the standard buffer conditions used in enzymatic DNA sequencing, PCR or LCR and enzyme will be inhibited by the reaction products.
Thus, there is a need for a thermostable (ATP, Mg.sup.2+)-independent relaxing DNA topoisomerase that is not inhibited by single-stranded DNA, equally active on positively and negatively supercoiled DNA, active through a wide range of temperatures and a wide range of salt conditions, to allow the performance of these manipulations on DNA more conveniently and more efficiently.